Page 379 - Avian Virology: Current Research and Future Trends

370 | Tripathy

strains are similar, with a high proportion of comigrating frag- antibody responses, ELISA is the most convenient method of
ments, most strains could still be distinguished by the presence or choice. In this regard, Buscaglia (2016) evaluated the immune
absence of one or two DNA fragments. In this regard, the genomic response by a commercial vaccine against fowlpox virus and the
profiles of fowlpox, quailpox, canarypox, and mynahpox viruses lesion at the site of inoculation. It was concluded that there is a
are distinct. Similarly, the Hawaiian bird pox viruses, alalapox and relationship between the ELISA values to the fowlpox vaccine
apapanepox, have genetic differences that distinguish them from that are considered positive and the presence of post-vaccination
each other as well as from FWPV (Kim and Tripathy, 2006a). In lesions. Although the agar-gel precipitation test is very simple,
an attempt to identify and differentiate vaccine and field strains easy to perform and convenient, its sensitivity is lower than
of FWPV, Tadese and Reed (2003) used RFLP, immunoblotting ELISA. However, it is still being used where facilities for ELISA
and PCR amplification of 4b gene. may not be available.

Polymerase chain reaction (PCR)
Any gene fragment of avian pox virus can be amplified by using Immunoblotting
specific primers designed from the available sequences of fowl- Comparisons of immunogenic proteins of strains of fowlpox by
pox and canarypox virus. Since all avian pox viruses produce immunoblotting, common as well as unique antigens are detected
A-type inclusion bodies, we have frequently used primers for (Schnitzlein et al., 1988) with polyclonal antibody. Similarly,
amplification of this gene. Similarly, analysis of a 578 bp PCR differences in antigenic profiles of fowlpox and other avian pox
amplified P4b gene fragment of FWPV, which is highly con- viruses are observed by using polyclonal anti-fowlpox or anti-
served, has been used frequently to discriminate among avian quailpox virus antibody (Ghildyal et al., 1989; Kim and Tripathy,
pox viruses. PCR analysis of the P4b gene was initially used as a 2006a).
diagnostic marker for FWPV infections. Phylogenetic relation- When two FWPV-specific monoclonal antibodies, P1D9 and
ships of avipoxviruses analysed based on the gene corresponding P2D4 were evaluated by Western blotting for antigenic charac-
to P4b shows that all avipoxvirus strains cluster into three major terization of 11 FWPV field isolates, 6 FWPV vaccine strains, and
clades, i.e. A (FWPV-like), B (CNPV-like) and C (Psittacinepox 3 pigeonpox virus vaccines, differences were observed. Whereas,
virus-like) (Jarmin et al., 2006). Further, subclades within the monoclonal antibody (mAb) P2D4 consistently recognized
clades have been observed. This approach has been used in a protein with an apparent molecular weight of 60 kDa in all
many cases for phylogenetic characterization, e.g. Offerman et vaccine and field strains, there was variability in the size of the
al. (2013) observed phylogenetic and histological variations in antigen that was immunoreactive with the other mAb P1D9. It
avian pox viruses isolated in South Africa. Several conserved recognized an antigen of apparent molecular weight of 46 kDa in
regions of the genome and gross and histopathological CAM all vaccine strains except two of FWPV origin. In these excep-
lesions were evaluated for this purpose. Rampin et al. (2007) tions, either only a 39-kDa or both a 42- and 46-kDa protein
confirmed the infection in buzzards by sequence analysis of 4b were immunoreactive. As for the field isolates, a 39-kDa antigen
gene to indicate the infection was due to subclade A2. In addi- was recognized in eight of them, whereas a 42-kDa antigen was
tion to 4b gene, DNA polymerase gene was used by Gyuranecz detected in the remaining three (Singh et al., 2003). These mAbs,
et al. (2013) for phylogenetic discrimination of 111 poxvirus iso- however, did not show any reaction with avian pox virus isolates
lates. All available sequences from GenBank were also included. from endangered Hawaiian forest birds (Kim and Tripathy,
When avipoxviruses causing diphtheritic and cutaneous lesions 2006a).
in Magellanic penguins in Brazil were characterized by analysis While both humoral and cell-mediated immune (CMI)
of P4b gene (Niemeyer et al., 2013), two different virus strains responses are important for protection against poxvirus infec-
clustering in clade A and B with differences in virulence were tions, CMI responses are seldom measured. In a study where both
observed. responses were measured, high levels of anti-FWPV antibodies
Based upon 538 bp fragment analysis of 4b gene, Jarvi et al. were detected by ELISA. Seroreactive polypeptides (B-cell anti-
(2008) identified two distinct variant clusters of poxviruses infect- gens) of FWPV antigen with molecular weights of 44.5, 66.5, 75,
ing Hawaiian forest birds. Poxvirus isolates from one of these two 90.5, and 99 kDa were detected by Western blot analysis. Also,
clusters appeared closely related to canarypox and other passerine significant increases in CMI responses were observed in inocu-
pox viruses, while the second appeared more specific to Hawaii. lated chickens as determined by lymphocyte proliferation assay,
cytotoxicity assay, and T-cell immunoblotting. The predominant
T-cell antigen of FWPV detected had a molecular weight of
Immune responses 66.5 kDa (Roy et al., 2015).
Avian pox viruses induce both humoral and cell-mediated Based upon information obtained from genetic studies (RFLP
immune responses. Antibody responses against natural fowlpox comparisons, PCR and nucleotide sequence analysis), antigenic
virus infection or a successful vaccination can be determined by studies (immunoblotting with polyclonal and selected monoclo-
serology, demonstrating anti-viral antibody. In this regard, a pas- nal antibodies) and biological studies (pathogenesis in chickens
sive haemagglutination, immuno-diffusion, virus neutralization, and challenge studies), it is clear that genetically, antigenically
immuno-fluorescence and enzyme linked immuno-sorbent assay and biologically different strains of pox viruses infect domestic,
(ELISA) have been used. However, for evaluation of humoral pet, and wild birds.